The nuclear pore complex (NPC) mediates the specific, bi-directional transport of large protein and RNA molecules across the nuclear envelope of a eukaryotic cell. Consequently, proper NPC function is essential for a wide variety of cellular biosynthetic and regulatory processes. Small perturbations of transport function can cause disease; for example, inefficient or excess transport of a single gene regulatory factor can give rise to cancer. The goal of the proposed research is to fundamentally advance our knowledge of NPC function by using in vitro single-molecule biophysics techniques to directly observe the NPC-mediated import of individual protein molecules bearing a nuclear localization signal and tagged with a fluorescent marker. The specific aims of the project are: 1) to identify spatially distinguishable intermediates in the transport process by tracking the nanometer-scale movement of individual, labeled substrate molecules as they travel across the nuclear envelope of isolated frog oocyte nuclei; and 2) to define a minimal kinetic mechanism for substrate transport by following the sequence of intermediates through which individual substrate molecules pass and determining the characteristic lifetimes of the intermediate states.